Influenza HA in SHIV VLPS for mucosal vaccination

描述（由申请人提供）：最近，我们利用杆状病毒表达系统成功生产了含有SIV Gag和HIV Env的猿人免疫缺陷病毒样颗粒（SHIV VLPs）。此外，我们还将流感病毒表面糖蛋白血凝素（HA）加入到SHIV VLPs中。利用透明质酸与上呼吸道粘膜的高亲和力，我们的中心假设是使用含有流感透明质酸的SHIV VLPs作为粘膜疫苗将增强对HIV感染的全身和粘膜免疫反应。本项目的主要重点是在小鼠模型中研究HA在SHIV VLPs中用于鼻内免疫的内建佐剂的效率和机制。具体而言，我们建议：1)。目的：确定与流感HA结合的SHIV VLPs作为一种粘膜疫苗在增强抗HIV免疫应答中的作用。拟进行的实验将探讨：a)在SHIV VLPs中，流感HA作为粘膜疫苗的内建佐剂是否比可溶性流感HA在增强全身和粘膜免疫方面更有效；b)流感HA的受体结合或膜融合活性是否影响其对SHIV VLPs的佐剂性。2）. 确定SHIV VLPs与流感HA结合在树突状细胞（DC）结合、激活、细胞因子产生和抗原呈递中的作用。我们建议研究：a) HA/SHIV VLPs是否具有增强的DC结合、内化和亚细胞定位能力；b) HA/SHIV VLPs对DC激活和细胞因子产生的影响是否增加；c). HA/SHIV vlp活化的dc是否增加初始T细胞增殖；d) HA/SHIV VLPs是否增加dc对CD8+ T细胞的抗原交叉呈递效率，以及相关的细胞内途径是什么。3）. 在没有CD4+ T细胞帮助的情况下，确定SHIV VLPs中与流感HA结合在特异性B细胞结合、激活和抗体产生中的作用。实验旨在研究：a) HA/SHIV vlp是否增加其与幼稚B细胞的结合能力；b). HA/SHIV VLPs对初代b细胞增殖的影响是否增加；c) HA/SHIV vlp激活的dc是否具有增强的粘附幼稚B细胞的能力；d). HA/SHIV vlp激活的dc是否增加初始B细胞的增殖；e) CD4+ t细胞缺陷小鼠经HA/SHIV VLPs鼻内免疫诱导位点鼻相关淋巴组织（NALT）是否发生B细胞活化、分化和细胞毒性CD8+形成。该项目是开发有效和安全的艾滋病毒疫苗的一种新方法。了解小鼠体内HA/SHIV vlp增强免疫反应的细胞和分子机制，对于未来在非人类灵长类动物模型和人体试验中设计和测试成功的HIV疫苗至关重要。

DESCRIPTION (provided by applicant): Recently, we have successfully produced simian-human immunodeficiency virus-like particles (SHIV VLPs) which contain SIV Gag and HIV Env by using a baculovirus expression system. Furthermore, we have incorporated the influenza virus surface glycoprotein, hemagglutinin (HA), into SHIV VLPs. Taking advantage of HA having a high affinity to bind to the mucosa of the upper respiratory track, our central hypothesis is that use of SHIV VLPs containing influenza HA as a mucosal vaccine will enhance both systemic and mucosal immune responses against HIV infection. The major focus of this project is to investigate the efficiency and mechanisms of the built-in adjuvanticity of HA in SHIV VLPs for intranasal immunization in a mouse model. Specifically, we propose: 1). To determine the role of incorporation with influenza HA in SHIV VLPs as a mucosal vaccine in enhancement of immune responses against HIV. Proposed experiments will investigate: a) whether the built-in adjuvanticity of influenza HA in SHIV VLPs as a mucosal vaccine is more potent than soluble influenza HA in enhancement of both systemic and mucosal immunity; and b). whether the receptor binding or membrane fusion activity of influenza HA affects its adjuvanticity for SHIV VLPs. 2). To determine the role of incorporation with influenza HA in SHIV VLPs in dendritic cell (DC) binding, activation, cytokine production, and antigen presentation. We propose to investigate: a). whether HA/SHIV VLPs have an increased ability to DC binding, internalization, and subcellular localization; b). whether HA/SHIV VLPs have an increased effect on DC activation, and cytokine production; c). whether HA/SHIV VLP-activated DCs increase naive T cell proliferation; and d). whether HA/SHIV VLPs increase the efficiency of antigen cross-presentation of DCs to CD8+ T cells and what are the associated intracellular pathways. 3). To determine the role of incorporation with influenza HA in SHIV VLPs in specific B cell binding, activation, and antibody production without CD4+ T cell help. Experiments are designed to investigate: a). whether HA/SHIV VLPs increase their ability to bind to naive B cells; b). whether HA/SHIV VLPs have an increased effect on naive B cell proliferation; c). whether HA/SHIV VLP-activated DCs have an increased ability to adhere to naive B cells; d). whether HA/SHIV VLP-activated DCs increase naive B cell proliferation; and e). whether B cell activation and differentiation and cytotoxic CD8+ formation occur in nasal-associated lymphoid tissue (NALT), an inductive site after intranasal immunization with HA/SHIV VLPs in CD4+ T-cell-deficient mice. This project represents a novel approach to develop an effective and safe HIV vaccine. Understanding the cellular and molecular mechanisms of HA/SHIV VLP-enhanced immune responses in mice is critical for the future design and testing of a successful HIV vaccine in non-human primate models and in human trials.